Lecture 9- Mechanisms

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  • Quiz 9 is available

  • Homework 4 assigned

Enzyme Mechanisms and Kinetics

Conformational States of Dimeric Proteins

  • Dimeric proteins can exist in two conformational states at equilibrium.

  • Conformational states interact with substrates and affect enzyme activity.

  • Equilibrium Shift: Substrate binding shifts equilibrium in favor of the active conformation.

Enzyme-Substrate Complexes

  • Representation:

    • E + S ↔ ES (enzyme-substrate complex)

    • E + P (enzyme-product complex)

  • Kinetic parameters define the rate of these transitions.

  • Various states related to substrate binding and product formation.

Kinetics of Chemical Reactions

Reaction Rate Changes

  • Initial Velocity: The slope of the progress curve at the beginning of a reaction correlates with substrate concentration.

  • Impact of initial substrate concentration on rate.

Rate Constant

  • The slope in initial velocity vs. substrate concentration is the rate constant (k).

Michaelis-Menten Kinetics

Michaelis Constant (Km)

  • Km is the substrate concentration at which the reaction velocity is half of Vmax.

  • Km characterized by a rectangular hyperbola in the velocity substrate concentration plot.

  • Formula:

    • ( v_0 = \frac{V_{max}[S]}{K_m + [S]} )

Lineweaver-Burk Plot

  • Double reciprocal plot derived from the Michaelis-Menten equation.

  • Provides a linear representation of enzyme kinetics:

    • ( \frac{1}{v_0} = \frac{K_m}{V_{max}} \frac{1}{[S]} + \frac{1}{V_{max}} )

Bi-substrate Reactions

Sequential Reactions

  • All substrates must bind before any product is released.

  • Substrate binding may be ordered or random.

Ping-Pong Mechanism

  • One substrate binds, and product is released while the enzyme retains a modified state before the second substrate binds.

Enzyme Inhibition

Types of Inhibition

  • Reversible Inhibition: Competitive, uncompetitive, and non-competitive.

  • Irreversible Inhibition: Causes permanent loss of enzyme activity.

Competitive Inhibition

  • Inhibitor competes with substrate at the active site.

  • Effect on kinetics: Vmax remains constant, Km increases (indicating decreased affinity).

Uncompetitive Inhibition

  • Inhibitor binds only to the enzyme-substrate complex.

  • Effect on kinetics: both Vmax and Km decrease.

Non-competitive Inhibition

  • Inhibitor can bind to either enzyme or enzyme-substrate complex.

  • Effect on kinetics: Vmax decreases while Km remains unchanged.

Catalytic Triad of Serine Proteases

  • Comprised of three amino acids: Serine, Histidine, Aspartate.

  • Each plays a distinct role in the catalytic mechanism:

    • Serine: Acts as a nucleophile.

    • Histidine: Acts as an acid-base catalyst.

    • Aspartate: Stabilizes the histidine residue.

Enzyme Regulation

Role of Allosteric Enzymes

  • Allosteric effectors (activators and inhibitors) bind non-covalently and can alter Km or Vmax.

  • Cooperativity: The presence of multiple subunits enhances the enzyme's response to substrate concentration changes.

Covalent Modification

  • Various covalent modifications (phosphorylation, acetylation) can activate or deactivate enzymes.

  • Example: Regulation of pyruvate dehydrogenase through phosphorylation.

Cofactors and Coenzymes

Cofactors

  • Essential ions and coenzymes necessary for enzyme function.

  • Differentiate between the strength of interaction with their apoenzymes.

Coenzymes vs. Vitamins

  • Coenzymes: Add functionalities not present in amino acid side chains.

  • Vitamins: Nutritional requirements where some are converted into coenzymes.

Major Coenzymes

  • Table highlights vitamins and their associated deficiency diseases, e.g., Vitamin C (ascorbate) prevents Scurvy.

Redox Coenzymes

  • Examples include NAD+/NADH and FAD/FADH2 involved in oxidation-reduction reactions.

Group Transfer Coenzymes

  • Key coenzymes: ATP (energy transfer), SAM (methyl transfer), and Coenzyme A (acyl transfer functionality).

Glycolysis and Cancer Metabolism

Warburg Effect

  • Cancer cells prefer aerobic glycolysis even in the presence of oxygen, leading to rapid glucose consumption.

  • Inefficient energy production with high glucose turnover raises questions about metabolic needs of tumor cells.